The goals of this investigation are to explore and define the relationship of the cardiovirus genus to other members of the picornavirus family and to exploit the unique features of cardioviruses to examine fundamental molecular questions about picornavirus translation, proteolytic processing, morphogenesis and host interaction. The RNA picornaviruses are one of the best understood and most thoroughly accessible experimental systems in all of biology. Natural infections with cardioviruses, like encephalomyocarditis virus (EMCV), kill nearly every cell in the brain, pancreas and heart of a mouse, within 3 days. The virus does this with apparent impunity to cellular antiviral defenses by subverting innate immunity traps and crippling the capacity of an infected cell to mount a defense or trigger an alarm. The molecular battleground inside infected cells pits viral protease 3Cpro and two unique cardiovirus proteins, L and 2A, enzymes honed by evolution for their special anti-cellular purposes, against the complete array of innate host defenses. The outcome rarely varies. Within 2-3 hours of infection EMCV brings to a halt cellular mRNA transcription, cap-dependent mRNA translation, antiviral signal transduction, and active protein/RNA exchange between the nucleus and cytoplasm. The virus replicates with fecundity and the cell dies before it ever triggers an alarm. At the ultimate molecular level, the activities of these proteins instigate the cascade of events that set off or prevent an episode of disease. The next phase of this project will examine the biochemistry and molecular pathways of EMCV L (Leader), the first viral (or cellular) protein known to bind and inactivate Ran GTPase cycling, the crucial, ubiquitous regulatory system for all protein and nucleic acid trafficking into and out of the nucleus. The project also examines the pathways by which the presence of L, or L-Ran complexes activate a specific cohort of cellular kinases, and redirects them towards the phosphorylation of nuclear pore proteins (Nups). The specific aims are: (1) To resolve the NMR structure of Mengo L (Leader) protein as it interacts with Ran GTPase. (2) To characterize the biochemistry of cardiovirus L:Ran interactions which inhibit RanGDP/GTP cycling in cell-free extracts. (3) To identify within cells, the host kinases activated by L, which contribute to the abrogation of nucleocytoplasmic trafficking steps. (4) To define the molecular interactions of L with other viral proteins (2A and 3CD), and define the replication advantages to cardioviruses, for encoding a unique and potent inhibitor of Ran and of cellular protein and mRNA trafficking. These objectives build directly upon experimental foundations developed during the preceding 27 years of the program. PUBLIC HEALTH RELEVANCE: It is rare to establish productive viral infections that lead to disease if the first infected cells or host immune system are able to fight back effectively. In a war won by stealth, an inadvertant triggering of intracellular or extracellular immunological alarms usually spells disaster (and clearance) for the virus. It's the job of the first viral proteins produced in the first 2-3 hrs of infection, to shutoff essential host response systems. This project examines the molecular pathways for how this happens with RNA picornaviruses in the cardiovirus genus.